Polyketide natural products from filamentous fungi are highly diverse in both chemical structures and bioactivities, and they include the current top-selling drugs such as lovastatin (for cholesterol lowering), as well as potent toxins such as aflatoxin and cercosporin. Polyketides are biosynthesized by a multi- enzyme complex called polyketide synthase (PKS). There is a knowledge gap in correlating the PKS structures with protein-ligand interactions, enzyme catalysis, and substrate specificity. Such a knowledge gap has severely hampered our efforts to biosynthesize new polyketide-based therapeutics by PKS engineering. To address this issue, we aim to solve the crystal structures of non-reducing PKS (NRPKS), to correlate the product outcome with protein structures, and to biosynthesize new polyketides based on the structure-function studies. We will determine the sequence-structure-function relationship of the NRPKS complex and two NRPKS domains, the starter unit:ACP transacylase (SAT) and product template (PT). SAT and PT catalyze the polyketide chain initiation and cyclization, respectively, in a highly specific manner. We will pursue the following specific aims: Aim 1. Determine the molecular basis of cyclization specificity in NRPKS by crystal structures and mutagenesis different PTs that will synthesize new polyketides with altered cyclization patterns. Aim 2. Determine the molecular basis of starter unit specificity in NRPKS by crystal structures and mutagenesis different SATs followed by combinatorial biosynthesis to yield new polyketides with different starter units and cyclization patterns. Aim 3. Determine the importance of protein-protein interaction on product outcome using chemical crosslinkers by specific cross-linking probes that stabilize the complex and facilitate crystallization of multi-domain PKS complexes. We have already obtained diffracting crystals of crosslinked, multi-domain PKSs (a first in the PKS field), crystal structures of PTs, diffracting crystals of PTs and SATs conveying different specificities, validated crosslinkers, and optimized enzyme assays. Outcomes from the proposed research have two aspects of potential overall biomedical impact: (1) new polyketides with different cyclization patterns and starter units may be screened for new bioactivities, (2) the structures of PTs, SATs and NRPKS complex that biologically produces toxins can be applied to structure-based inhibitor design to identify new chemo-preventative agents against fungal toxin biosynthesis. Outcomes from the proposed research will have a high overall scientific impact, because it will not only determine how fungal PKSs specifically cyclize (AIM 1) and initiate (AIM 2) polyketide biosynthesis, but will also result in the first crystal structure of a PKS complex and elucidate how protein-protein interactions in the mega-synthase affects the product outcome (AIM 3).